Objectives The aim of this study was to explore the clinically relevant situation of tethering and MI, testing the hypothesis that ischemic milieu modifies mitral valve adaptation.

Methods Twenty-three adult sheep were examined. Under cardiopulmonary bypass, the papillary muscle tips in 6 sheep were retracted apically to replicate tethering, short of producing MR (tethered alone). Papillary muscle retraction was combined with apical MI created by coronary ligation in another 6 sheep (tethered plus MI), and left ventricular remodeling was limited by external constraint in 5 additional sheep (left ventricular constraint). Six sham-operated sheep were control subjects. Diastolic mitral valve surface area was quantified by 3-dimensional echocardiography at baseline and after 58 ± 5 days, followed by histopathology and flow cytometry of excised leaflets.

As the left ventricle remodels, the surface area of the stretched MV increases adaptively to reduce MR (4–6). Ischemic MR remains common, however, indicating inadequate leaflet compensation (4,5). Valves excised at the late heart failure stage are stiff and fibrotic (7–9), further impairing closure by reducing systolic leaflet expansion and the flexibility needed to bend and seal effectively, as shown by finite-element analysis (10). Intrinsic MV changes are therefore important for the full pathogenesis of ischemic MR and its persistence following annuloplasty (11–13).

Valve adaptation can be affected not only by mechanical stretch but also by the ischemic milieu, with its known cytokine release (14–16), and by MR turbulence (17,18). We developed a large-animal model to vary these factors independently, following MV area noninvasively by 3-dimensional echocardiography (Figures 1B, 1C, 1E, and 1F) (4,5,19).

How the tethered valve is affected by ischemia, with release of inflammatory cytokines and transforming growth factor (TGF)-β (14–16,24–27), is unknown. We therefore tested the hypothesis that MI alters adaptation of tethered MVs. A limited apical MI was used to avoid direct PM displacement by inferior MI (Figure 1D) (28). MV area changes over 2 months were explored by excised-leaflet molecular histopathology and flow cytometric analysis.

The tethered plus MI group required controlled leaflet tethering without MR or leaflet disruption, achieved after left thoracotomy and pericardial cradle construction under cardiopulmonary bypass. The left atrium was opened, and suture loops were inserted via the MV orifice into the exposed PM tips (both medial PM heads), buttressed by Teflon felt pledgets, and exteriorized to the epicardium overlying the PMs, pulled through a Dacron anchoring patch, in a model developed by J. Luis Guerrero. Retracting these sutures parallel to the PM axis pulled the PM tips and leaflets apically (19). The heart was restarted and a limited apical infarct produced by distal left anterior descending coronary artery ligation, avoiding directly increased PM tethering from inferior wall bulging (28). Final suture length was then adjusted in the beating heart under echocardiographic guidance just short of producing MR, the sutures were knotted against the anchoring patch, and the chest was closed.

To limit post-infarction LV remodeling and its potential effects on leaflet tethering and MV adaptation, the tethered plus MI with LV constraint group required suturing a flexible Prolene surgical mesh (Ethicon, Somerville, New Jersey) circumferentially from base to apex around both ventricles after the creation of the same MV tethering and MI model (3). This allowed us to explore MV adaptation in an ischemic milieu with limited post-infarction LV dilation.

All sheep underwent cardiopulmonary bypass, with no MI created in the tethered-alone group and no surgical intervention in the control subjects after MV and PM exposure.

Animals were cared for 58 ± 5 days and euthanized after left thoracotomy with echocardiography to confirm absence of MR and reconstruct MV surface area. These studies conformed to National Institutes of Health animal care guidelines and had institutional animal care approval.

Imaging and analysis

Echocardiographic data were collected using epicardial high-frequency (3.5- to 5-MHz) 2-dimensional and 3-dimensional echocardiographic probes (S5 and X3) with an iE33 scanner (Philips Medical Systems, Andover, Massachusetts). Three-dimensional volumetric datasets were ECG gated from 4 to 7 consecutive heartbeats. Full datasets were acquired in standardized planes at baseline and before euthanasia. MR absence or grading by long-axis-view vena contracta and successful MV leaflet tethering were assessed in the beating heart just after PM tethering and MI creation and again prior to euthanasia. Three-dimensional LV volumes were calculated from 6 equiangular rotated apical views. Three-dimensional tenting volume was measured between the closed leaflet atrial surface and the annular least squares plane (Figures 1C and 1F) (3).

Total MV leaflet area was measured at full diastolic opening to factor out superimposed passive systolic stretch. (Total leaflet area cannot be measured precisely in systole, because the coapted portions cannot be optimally resolved). Leaflet area was measured in a blinded fashion using validated custom Omni4D software (M. D. Handschumacher, Boston, Massachusetts) (Online Appendix) (4,29).

The left atrium was opened and the LV wall dissected from the anterolateral commissure under irrigation of pre-cooled sterile phosphate-buffered saline. Both leaflets including chordae were divided for histopathology (frozen in optimal cutting temperature compound at −80°C) (Online Appendix) and cell isolation and flow cytometry (transported in pre-cooled physiological collecting medium) (Online Appendix).

Microscopy was used to measure leaflet thickness in the 10 thickest areas across the leaflet midportion and anterior and posterior strut chordal thickness.

Statistical analysis

One-way analysis of variance with post-hoc contrast analysis and Tukey-Kramer test were used when appropriate to compare multiple groups of interest. Paired Student t tests were used to compare baseline and euthanasia results within animals. Data are summarized as mean ± SD for continuous variables and as medians with percentiles as appropriate. Statistical significance was set at p < 0.05 (2-sided).

(A) Staining for interstitial marker α–smooth muscle actin (SMA) along the CD31-positive endothelium shows increasing extension of α-SMA-positive cells into the interstitium (asterisks) for the study groups with tethered mitral valves; occasional CD31-positive staining was seen in the interstitium. (B) Quantitative analysis of endothelial cells (ECs) detected by flow cytometry that are also α-SMA-positive, indicating endothelial-to-mesenchymal transformation (EMT), and expressed as percentage of total CD31-positive cells. Abbreviations as in Figure 1.

Increased VCAM-1 along the endothelium could increase cellular recruitment into the valve. We therefore evaluated MV sections for cells expressing the hematopoietic marker CD45. Tethered plus MI MVs contained significantly more CD45-positive cells, uncommon in tethered-alone MVs and control MVs (Figures 5A and 5B). Cell proliferation detected by Ki67 immunostaining was also strongly increased in both endothelial and interstitial cells of tethered plus MI MVs (Figures 5C and 5D). CD45- and Ki67-positive cells were predominantly in the atrialis and spongiosa.

Ingrowth and remodeling

Normal cardiac valves are largely avascular. Neovascularization is common in degenerative disease and potentially facilitates inflammation and cell recruitment. Tethered plus MI valves showed more microvessels in their midportions than tethered-alone and control MVs (Figures 5C and 5D).

LV constraint

Three-dimensional echocardiographic analyses (Online Appendix) showed that tethered plus MI sheep had increased LV volumes and tethering distances at euthanasia compared with tethered-alone sheep. To explore whether the MV changes also occur without such increases in volume, we studied 5 additional tethered plus MI sheep in which LV volumes were limited by external constraint. With this constraint, LV volumes and tethering distances at euthanasia were comparable with those with tethering alone and significantly less than in tethered plus MI sheep without constraint (Online Appendix, Online Figure 4). Despite this reduction in LV expansion and tethering, MV changes were qualitatively and quantitatively comparable with those in tethered plus MI sheep without constraint, including consistently and prominently increased leaflet thickness (Figures 2A and 2B), EMT with extensive subendothelial α-SMA staining (Figures 3A and 3B), TGF-β and endothelial VCAM-1 staining (Figure 4A), CD45-positive cells (Figures 5A and 5B), Ki67-positive cells (Figures 5C and 5D), microvasculature ingrowth (Figures 5C and 5D), and MMP-2/MMP-9 (Figure 6). Leaflet area increased less in the constrained tethered plus MI sheep, consistent with the smaller left ventricle (Figure 2D) and potentially corresponding to the mildly but not significantly lower percentage of endothelial cells undergoing EMT and Ki67-positive cells (Figures 3B and 5D). Chordal thickness, EMT, and CD45-positive cells were also comparable (Figure 7).

Discussion

This study goes beyond prior in vivo observations that mechanical MV tethering reactivates EMT, an embryonic growth process, increasing both leaflet area and thickness (19,21–23). Stretch induces valve expression of TGF-β (31), an EMT promoter (32). TGF-β was expressed but not strongly with tethering alone, suggesting that mild TGF-β induction may suffice to promote leaflet growth or that other signals may also activate EMT. These findings indicate that adult valves, normally quiescent (20), can respond to stress and altered ventricular geometry (19,20,23), as seen with their enlargement in the remodeled ventricles of patients with aortic insufficiency (33).

As expected, tethering is mildly augmented by MI-induced LV remodeling, and this may contribute to changes in the MV. However, MI-induced valve changes persist when external constraint limits LV remodeling and reduces tethering distances. Furthermore, tethered-alone and tethered plus MI valves are different not only in degree of EMT, thickness, and TGF-β expression but also in nature of changes, with endothelial VCAM-1 activation, accumulation of CD45-positive cells, and neovascularization. The post-MI effects on MV adaptation therefore do not appear to depend solely on LV remodeling, although it seems reasonable that they may increase with LV remodeling-induced tethering.

For example, TGF-β, profibrotic in many organs, is overexpressed in the tethered plus MI valves and seen in areas of increased collagen deposition (Figures 4A and 4B). Although TGF-β is a key stimulus for EMT and growth in the developing valve (21–23), it could drive valve fibrosis by activating valvular interstitial cells to become contractile α-SMA-positive myofibroblasts that secrete, compact, and remodel the extracellular matrix (24–27,35,36). The fibrogenic effects of TGF-β might be amplified by its activation of angiotensin type–1 receptors (26) that in turn would increase TGF-β expression.

TGF-β as a compensatory response to stretch may therefore become counterproductive post-MI (30), causing excessive EMT as a substrate for valve fibrosis, stiffening, and regurgitation (8–10,35). TGF-β may further lead to myofibroblast-induced matrix contraction (25) that can limit the ability of the valve to increase its area, possibly explaining the relative deficiency in valve area post-MI despite increased ventricular size (Central Illustration). TGF-β can also drive the increased chordal EMT and fibrotic thickening seen in the tethered plus MI valves (Figure 7), further restricting leaflet closure and increasing regurgitation.

TGF-β may originate from the stretched valve itself; TGF-β is also markedly increased in ischemic myocardium and released into the cardiac extracellular fluid (24). Conceivably, post-MI neurohumoral activation of profibrotic angiotensin II and aldosterone may promote fibrosis, synergistically with TGF-β.

An unexpected finding was the prominence of cells positive for CD45, considered a pan-hematopoietic cell marker, in the tethered plus MI leaflets and chordae (Figures 5A, 5B, and 7). Although these cells require further characterization, one hypothesis consistent with other findings is that they are fibrocytes, circulating bone marrow–derived cells that enter sites of injury and inflammation and adopt an α-SMA-positive myofibroblast phenotype (38–41). Fibrocytes, beneficial in normal wound healing and compaction, produce stiffened, sclerotic tissue in pulmonary fibrosis, asthma, and fibrosing renal and cutaneous diseases (39–41). They are also recruited to ischemic myocardium, where their inhibition reportedly reduces cardiac remodeling (42).

An equilibrium normally exists between circulating bone marrow–derived cells and the cardiac valves (43); this may be altered by activating cell adhesion molecules (37), as in the tethered plus MI valves. Marrow-derived cell recruitment is a ubiquitous response to diverse vascular and valvular injuries. Tanaka et al. (44) showed that marrow-derived cells recruited to the aortic valve display a mesenchymal phenotype in hypercholesterolemic mice.

Clinical and therapeutic implications

The observations in this study suggest post-MI transition from adaptive MV enlargement to maladaptive limitation of enlargement and counterproductive stiffening (15,26). TGF-β overexpression, exuberant EMT as substrate for fibrosis, and endothelial activation with circulating cell recruitment in the fibrocyte hypothesis can promote fibrosis and matrix compaction (Central Illustration).

Understanding these mechanisms has potential therapeutic implications, with the long-range goal of augmenting MV area and preserving leaflet flexibility to reduce ischemic MR. TGF-β pathways can be inhibited, for example, using angiotensin type–1 receptor blockers as TGF-β antagonists to limit tissue growth in a Marfan mouse model (47). Pharmacological modification may require a specific time window to permit early EMT but prevent later fibrosis (24).

Study limitations and future directions

We compared changes when MI was superimposed on mechanical tethering without MR. Further work will be needed to explore the effects of MI alone, MR turbulence, and TGF-β blockade. MR alone increases MV collagen deposition, MMPs, and circulating tumor necrosis factor–α (18,48) but down-regulates myocardial noncollagen ECM components and TGF-β (49); MR combined with tethering can be tested in inferior MI. Inferior MI increases valve area, but insufficiently to prevent MR (5). Whether CD45-positive cells are beneficial or adverse needs to be explored (43,46), as do the roles of VCAM-1 and TGF-β in cell recruitment.

To determine why cellular changes (EMT, VCAM-1) mainly affect the atrial side of the MV requires testing biological differences between atrial and ventricular endothelium, compounding the higher atrialis radius of curvature and stress. Longitudinal studies are needed to explore clinically important questions of how the MV changes over time, why leaflet adaptation is frequently inadequate to prevent MR, and how this can be improved.

Conclusions

MV adaptation to mechanical stretch created by PM tethering with concomitant MI is different in nature and extent than with PM tethering alone, including exuberant EMT, cellular proliferation, TGF-β overexpression, endothelial activation, accumulation of CD45-positive cells, neovascularization, and matrix remodeling. Potential explanations based on synergistic stretch- and MI-induced pathways involve TGF-β, proinflammatory cytokines, and recruited fibrocytes. Understanding these mechanisms could lead to new therapeutic opportunities to increase leaflet area and flexibility and preserve coaptation.

Perspectives

COMPETENCY IN MEDICAL KNOWLEDGE: A tethered MV has the potential for compensatory leaflet adaptation. In patients with volume overload from chronic aortic insufficiency, MV growth matches ventricular dilation, and MR is rare. With comparable ventricular dilation after MI, however, MV growth is often insufficient, and the leaflets become thick and fibrosed; MR is frequent, and heart failure and mortality risks are doubled.

TRANSLATIONAL OUTLOOK: Future research should seek to identify the cellular pathways through which post-MI tethering promotes MV leaflet and chordal growth so that therapeutic strategies can be developed on the basis of these adaptive mechanisms.

Appendix

Appendix

For a supplemental methods and results section as well as figures, please see the online version of this article.

Footnotes

This work was supported in part by grant 07CVD04 from Fondation Leducq (Paris, France) for the Transatlantic MITRAL Network of Excellence and by the National Heart, Lung, and Blood Institute of the National Institutes of Health under award number R01HL109506 to Drs. Levine, Aikawa, and Bischoff. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Additional support was from grant K24 HL67434 from the National Institutes of Health, an American Society of Echocardiography Career Development Award, and an Erwin-Schrödinger Stipend (FWF Austrian Science Fund). The biostatistical work was conducted with support from Harvard Catalyst, The Harvard Clinical and Translational Science Center (National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health award UL1 TR001102), and financial contributions from Harvard University and its affiliated academic health care centers. The content is solely the responsibility of the authors and does not necessarily represent the official views of Harvard Catalyst, Harvard University, and its affiliated academic health care centers or the National Institutes of Health. The authors have reported that they have no relationships relevant to the contents of this paper to disclose. Drs. Dal-Bianco, Aikawa, and Bischoff contributed equally to this work.

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